Group A streptococcal bacteremia: the role of tumor necrosis factor in shock and organ failure.

Severe group A streptococcal infections associated with early onset shock and multiorgan failure define the streptococcal toxic shock syndrome. In the United States, group A streptococcal strains most commonly isolated are M types 1 and 3, which produce pyrogenic exotoxin type A. The role of tumor necrosis factor (TNF)-alpha and the dynamics of cardiovascular and laboratory abnormalities were investigated in a baboon model of group A Streptococcal bacteremia that mimics human Streptococcal toxic shock syndrome. Profound hypotension, leukopenia, metabolic acidosis, renal impairment, thrombocytopenia, and disseminated coagulopathy developed within 3 h after intravenous infusion of M type 3, pyrogenic exotoxin A-producing group A streptococci. Serum TNF-alpha peaked at 3 h and returned to baseline by 10 h. Mortality was 100%. Anti-TNF-alpha monoclonal antibody treatment markedly improved mean arterial blood pressure, tissue perfusion, and survival, suggesting that TNF-alpha plays an important role in the induction of shock and organ failure in group A streptococcal bacteremia.

Effects of Clostridium perfringens recombinant and crude phospholipase C and theta-toxin on rabbit hemodynamic parameters.

Clostridium perfringens exotoxins have been implicated as major virulence factors responsible for shock and organ failure in gas gangrene, yet the mechanism(s) by which they mediate cardiovascular dysfunction remain enigmatic. Recombinant (r) phospholipase C (PLC), r theta-toxin, culture supernatant (crude toxin), or 0.9% NaCl was infused intravenously into awake rabbits. Cardiac index (CI), mean arterial pressure (MAP), heart rate (HR), central venous pressure (CVP), arterial blood gases, and hematocrit were measured 1 h before and for 3 h after toxin infusion. Crude toxin and rPLC decreased CI, MAP, and HR and increased CVP; mortality was 87.5% and 83%, respectively. r theta-toxin did not decrease CI or MAP and mortality was 25%. Further, crude toxin and rPLC but not r theta-toxin inhibited cardiac contractility (dF/dt) in isolated rabbit atrial muscles. These results suggest that PLC-induced myocardial dysfunction contributes to shock in C. perfringens infection.

Antibiotic effects on bacterial viability, toxin production, and host response.

The efficacy of an antibiotic in human or experimental infection is presumed to be proportional to its in vitro antimicrobial activity, yet antibiotics having comparable in vitro activity may have markedly different efficacies in vivo. For example, we have reported that clindamycin is more efficacious than penicillin in experimental gas gangrene caused by Clostridium perfringens in animals. To explain these differences, we compared the dynamics of bacterial killing and suppression of toxin synthesis. In addition, we investigated the ability of clindamycin and penicillin to modulate lipopolysaccharide-induced cytokine production in human peripheral blood mononuclear cells. Our results suggest that clindamycin affects protein synthesis in both prokaryotic and eukaryotic cells. These data may, in part, explain why the efficacy of clindamycin is greater than that of penicillin and demonstrate that clindamycin may be important immune modulator.

Investigation of the role of the disulphide bond in the activity and structure of staphylococcal enterotoxin C1.

The goal of this study was to investigate the role of the disulphide bond of staphylococcal enterotoxin C1 (SEC1) in the structure and activity of the toxin. Mutants unable to form a disulphide bond were generated by substituting alanine or serine for cysteine at positions 93 and/or 110. Although we did not directly investigate the residues between the disulphide linkage, tryptic lability showed that significant native structure in the cystine loop is preserved in the absence of covalent bonding between residues 93 and 110. Since no correlation was observed between the behaviour of these mutants with regard to toxin stability, emesis and T cell proliferation we conclude that SEC1-induced emesis and T cell proliferation are dependent on separate regions of the molecule. The disulphide bond itself is not an absolute requirement for either activity. However, conformation within or adjacent to the loop is important for emesis. Although mutants with alanine substitutions were not emetic, those with serine substitutions retained this activity, suggesting that the disulphide linkage stabilizes a crucial conformation but can be replaced by residues which hydrogen bond.

Predictions of T-cell receptor- and major histocompatibility complex-binding sites on staphylococcal enterotoxin C1.

We have focused on regions of staphylococcal enterotoxin C1 (SEC1) causing immunomodulation. N-terminal deletion mutants lacking residues 6 through 13 induced T-cell proliferation similar to that induced by native toxin. However, mutants with residues deleted between positions 19 and 33, although nonmitogenic themselves, were able to inhibit both SEC1-induced T-cell proliferation and binding of the native toxin to major histocompatibility complex (MHC) class II. Presumably, these deletions define a part of SEC1 that interacts with the T-cell receptor. Three synthetic peptides containing residues located in a region analogous to the alpha 5 groove of SEC3 had residual mitogenic activity or blocked T-cell proliferation induced by SEC1 and appear to recognize the same site as SEC1 on a receptor for the toxin, presumably MHC class II. We conclude that isolated portions of the SEC1 molecule can retain residual mitogenic activity but that the entire protein is needed to achieve maximal superantigenic stimulation. Our results, together with the results of other investigators, support a model in which SEC1 binds to an alpha helix of MHC class II through a central groove in the toxin and thereby promotes or stabilizes the interaction between antigen-presenting cells and T cells.

Superantigens associated with staphylococcal and streptococcal toxic shock syndrome are potent inducers of tumor necrosis factor-beta synthesis.

The role of tumor necrosis factor-alpha (TNF alpha) in the pathogenesis of severe bacterial infections has been studied extensively. However, the role of TNF beta, a lymphokine with biologic activities similar to those of TNF alpha, has received little attention. Therefore, the purpose of this study was to examine the production of TNF beta by peripheral blood mononuclear cells in response to lipopolysaccharide (LPS) and the superantigens staphylococcal toxic shock syndrome toxin 1 (TSST-1) and streptococcal pyrogenic exotoxin A (SPEA). Though LPS was a more potent inducer of TNF alpha than was TSST-1 or SPEA, TSST-1 and SPEA were both more potent inducers of TNF beta. The superantigens TSST-1 and SPEA were more potent inducers of total TNF (TNF alpha and TNF beta) than was LPS. These data suggest that the induction of TNF beta synthesis may be a unique pathway by which superantigens associated with severe streptococcal and staphylococcal infections mediate shock and multiorgan failure characteristic of toxic shock syndrome.

Streptococcal toxic shock syndrome: synthesis of tumor necrosis factor and interleukin-1 by monocytes stimulated with pyrogenic exotoxin A and streptolysin O.

Previous studies have found that 80% of strains isolated from patients with the streptococcal toxic shock syndrome produce pyrogenic exotoxin A (SPEA) and 100% produced streptolysin O (SLO). To elucidate the cellular mechanisms contributing to shock, human monocytes were stimulated with SPEA (0.1-10 micrograms/10(6) monocytes) or SLO (0.2-2.5 hemolytic units/10(6) monocytes), and production of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta was measured at 24, 48, and 72 h. SPEA and SLO were potent inducers of TNF alpha, with maximum production occurring at 72 h for SPEA and at 48 h for SLO (1067 and 687 pg/ml, respectively). In contrast, IL-1 beta production was greater for SLO than for SPEA (557 vs. 258 pg/ml). In addition, the effects of SPEA and SLO together were synergistic in terms of monocyte IL-1 beta production: SPEA, 193 pg/ml; SLO, 452 pg/ml; SPEA plus SLO, 799 pg/ml. These findings suggest TNF alpha and IL-1 beta are important candidates for mediating shock in severe streptococcal infections.

Nucleotide sequence of the staphylococcal enterotoxin C3 gene: sequence comparison of all three type C staphylococcal enterotoxins.

The structural gene entC3, which encodes staphylococcal enterotoxin C3 was cloned from the genome of Staphylococcus aureus FRI-913 and sequenced. The primary amino acid sequence of the toxin was deduced from the nucleotide sequence data. entC3 contains 801 bp and encodes a precursor protein of 266 amino acids. Glutamic acid was found to be the N-terminus of mature enterotoxin C3. Thus, the first 27 residues of the toxin precursor comprise the signal peptide, and the mature toxin contains 239 amino acids with a molecular weight of 27,563 daltons. Enterotoxin C3 differs from enterotoxin C2 by four amino acids and from enterotoxin C1 by nine residues. The 167 C-terminal residues of the three toxins are identical, except for one conservative amino acid substitution in enterotoxin C3. The degree of immunological relatedness among the three Type C enterotoxins is proportional to their molecular relatedness. This study also provides evidence that the N-termini of Type C enterotoxins determine subtype-specific antigenic epitopes, while more conserved C-terminal regions determine biological properties and cross-reactive antigenic epitopes shared with other pyrogenic toxins.